The ability to rapidly detect food and water-borne pathogens is of utmost importance in preventing outbreaks associated with contamination of our nation's food and water supply. Existing detection systems have a limited ability to simultaneously screen a single sample for multiple agents. To meet this need we propose to use the ligase detection reaction (LDR) combined with PCR, and Universal Array detection, which we have already validated in identifying and distinguishing blood-borne bacterial and viral pathogens, including Category A biothreat agents. We will transfer these assays onto the Cepheid GeneXpert system, as well as evaluate their performance in modular microfluidic devices. The above techniques will be used to meet the specific aims of this application: Aim 1. To identify multiple food and waterborne bacteria simultaneously from a single sample. We will develop multiplexed and nested PCR/LDR assays to identify Category B bacterial pathogens (Campylocbacter jejuni, Yersinia enterocolitica, Salmonella spp, Shigella spp, Diarrheagenic E. coli, Pathogenic Vibrio spp, and Listeria monocytogenes). Stool specimens from patients with diarrheal disease will be evaluated in the microbiology laboratory and tested using the above molecular techniques. Random samples will also be spiked with DNA of uncommon bacterial pathogens to simulate infection with these agents. Aim 2. To identify multiple food and waterborne protozoa and viruses simultaneously from a single sample. PCR/LDR and RT-PCR/LDR will be used to detect the Category B food and water-borne protozoa (Cryptosporidium parvum, Cyclospora cayatanensis, Entamoeba histolytica, Giardia lamblia and Microsporidia) and viruses (Caliciviruses, Hepatitis A virus and Rotavirus) respectively. Stool specimens from patients with suspected protozoan or viral infections will be evaluated in the microbiology laboratory and tested using these molecular techniques. Aim 3. To develop high throughput methods for screening of multiple food and waterborne pathogens using molecular signature profiles. Standardized protocols using common liquid handling robotic platforms will be established using the techniques developed in Aims 1 and 2. The test will be validated for detecting bacterial pathogens, protozoa, and viruses from 3,000 stool samples. The PCR/LDR assays will be migrated to the Cepheid GeneXpert system as well as other microfluidic devices initially using capillary electrophoresis or LDR-FRET, and subsequently Universal Array readout.